Signaling between cells can occur via receptor-receptor interactions for closely-opposed membranes or by transfer of soluble signaling molecules such as cytokines, such intercellular communications is especially key to the initiation of adaptive immune responses via the activation of T cells by antigen presenting cells (APCs). Recent studies have revealed the formation of an organized structure known as the immunological synapse between T cells and APCs during T cell activation, characterized by clustering of receptor pairs at the interface. Soluble cytokines are also known to be critical mediators of events during T cell activation, yet little is known about the transfer of these molecules from T cell to APC and vice versa during antigen recognition. To characterize cytokine transfer during T cell activation we will undertake the following specific aims: 1. We will develop a new technology aimed at detecting secreted cytokines near the surface of cells and within the immunological synapse as a means for characterizing in situ the temporal and spatial distribution of cytokines during T cell recognition via 3D time-lapse fluorescence imaging. 2. We will apply this approach to determining the spatial distribution of the key cytokine interleukin-2 (IL-2) near the surface of T cells and APCs during during T cell recognition as a function of time. 3. We will extend this technology to simultaneously characterize the spatial and temporal distribution of 3 cytokines believed to be exchanged between T cells and APCs during activation, IL-2, interleukin-12, and interferon-gamma (IFN-gamma). The proposed approach builds on existing enzyme-linked immunoabsorbent (ELISA) methods, but uses new technology for determination of local soluble molecule concentrations near the cell surface, using 3D fluorescence microscopy for direct spatially-resolved analysis. Results from these studies will 1) provide a new technology for characterizing soluble molecule transfer between cells, applicable to many biological systems, and 2) provide the first quantitative description of cytokine transfer between cells during T cell activation. Application of this new technology to this and other systems will allow the spatial and temporal complexity of cytokine signaling between cells to be directly characterized and thus allow rational therapies to be developed that target cytokine signaling.